The immunology , immunochemistry and cellular biology of two parasites that cause considerable disease in the United States are studied. Giardia lamblia is the most common disease-causing parasite in the United States and one of the earliest branching protist that possesses a unique secretory system. In more evolved cells, lysosomal proteins are sorted from the trans-Golgi network to endosomal/lysosomal compartments by inclusion into clathrin-coated vesicles. Lysosomal trafficking is achieved by specific cargo recognition involving adaptor proteins (AP) which recognize specific sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Earlier studies demonstrated that Giardia has a tyrosine-based sorting system, which mediates the targeting of encystation-specific cysteine protease (ESCP) to lysosome-like peripheral vacuoles (PVs). Studies over the past year show that the transport of two resident proteins, the membrane-associated ESCP and the soluble protein acid phosphatase (AcP), to the PVs is clathrin-adaptin dependent. By use of the yeast-two hybrid assay , we found that the ESCP tyrosine-based motif interacts specifically with Giardia AP-1 through interaction with its medium subunit (mu1). Using tagged-proteins in immunofluorescence analysis we were able to colocalize AP-1 with either ESCP or AcP. Pull-down assays of tagged proteins confirmed the ESCP/AP-1 interaction. Silencing of the Giardia-mu1 gene resulted in mislocalization of ESCP and AcP but not of variant-specific surface proteins (VSPs). Even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, proteins like VSPs are sorted and constitutively secreted to the plasma membrane, and cyst wall proteins (CWPs) are developmentally secreted in a regulated pathway. Moreover, the finding of an anterograde specific protein delivery to lysosome-like PVs by clathrin-adaptor proteins suggests that this parasite may possess a primitive secretory organelle capable of sorting proteins similar to more evolved cells. Our ability to transfect Giardia and to express or inhibt protein expression in vivo has allowed new approaches to understanding the cellular biology of Giardia. A number transfection vectors were developed. RNAi, a process and method where small RNA molecules are able to inhibit transcription and expression of proteins as well as cause silencing by chromatin alterations, has become an important tool to inhibit expression of proteins in some protozoa and helminths. Many times this approach allows us to determine the function of proteins usikng this approach. There is relatively good but as yet unpublished data(Lujan et al) that enzymes and mechanisms characteristic of RNAi are present in Giardia but its biological role is at present uncertain. Therefore inducible RNAi vectors were employed to inhibit protein expression. An RNAi vector was employed in the AP1 studies mentioned above and successfully inhibited transcription and expression of the AP1 protein. However, attempts to use this vector to inhibit expression of other proteins were not as successful. Another technical innovation was the modification of a vector capable of induction of proteins in vivo with tetracylcline( This vector was previously developed in another laboratory). This enabled the study of important or essential processes since consitutive expression would likely kill Giardia before selection. Another innovation was the development in our ability to express two proteins with unique epitope tags at the same time from the same vector. This enables co-localization and studies of potential interactions in vivo. That G. lamblia infection in mice deficient in Il-6 leads to prolonged infection and increased parasite burden was previously shown by Dr. Singer in this lab. Further studies demonstrated that Il-6 is important for control of acute infections but eventually infections are controlled. Lack of Il-6 did not prevent the production of specific IgA that in chronic infections reacted with the surface of most every parasite. The later suggests that immune mediated mechansims may be responsible to continued control of chronic subclinical infections in this model. Therefore there are multiple mechanisms to control infection in this model and there are clear differences in the mechanisms involved in the control of acute versus chronic disease. The second type of parasite studied is Microsporidia. These are intracellular eukaryotes that infect many animals and cause opportunistic infections in AIDS patients. The disease is transmitted via environmentally resistant spores. Cells are infected via a polar tube that infects cells by penetrating the host membrane and injecting its infectious sporoplasm into the host. Studies initiated here by Dr. J. Russell Hayman were further studied. One of the more common causes of microsporidiosis in humans is Encephalitozoon intestinalis. When E. intestinalis is cultured invitro, the spores adhere to the host cell surfaces. Although this adherence occurs spontaneously and can be viewed by light microscopy, it has not been described in detail. Studies were done to understand the mechanism of E. intestinalis spore adherence to host cells and to determine the association of spore adherence to infectivity. Since other eukaryotic microbes have been shown to exploit glycosaminoglycans (GAGs) in selection of and attachment to host cells, initial studies focused on the role of GAGs in E. intestinalis spore adherence to host cells. These studies revealed that sulfated GAGs are involved in spore-host cell adherence. The addition of exogenous GAGs to the adherence assay reduces E. intestinalis spores adherence to host cells by as much as 90%. In addition, treating host cells with compounds that either limits the expression of host cell surface GAGs (p-nitrophenyl-b-D-xylopyranoside) or limits the extent of host cell GAG sulfation (sodium chlorate) results in decreased spore adherence. Spore adherence assays using CHO mutant cell lines deficient in surface GAGs also indicate a significant decrease in adherent spores compared to the parent cell line. Taken together, these data implicate the involvement of host cell surface GAGs in the E. intestinalis spore adherence mechanism.